Metallothionein isoform 2A expression is inducible and protects against ROS-mediated cell death in rotenone-treated HeLa cells

The role of MT (metallothionein) gene expression was investigated in rotenone-treated HeLa cells to induce a deficiency of NADH:ubiquinone oxidoreductase (complex I). Complex I deficiency leads to a diversity of cellular consequences, including production of ROS (reactive oxygen species) and apoptosis. HeLa cells were titrated with rotenone, resulting in dose-dependent decrease in complex I activity and elevated ROS production at activities lower than 33%. Expression of MT2A (MT isoform 2A), but not MT1A or MT1B RNA, was significantly inducible by rotenone (up to 7-fold), t-BHP (t-butyl hydroperoxide; 5-fold) and CdCl2 (50-fold), but not ZnCl2. Myxothiazol treatment did not elevate either ROS or MT2A levels, which supports a ROS-related mechanism for rotenone-induced MT2A expression. To evaluate the role of MT2A expression, MT2A and MT1B were overexpressed in HeLa cells and treated with rotenone. Compared with control and MT1B-overexpressing cells, ROS production was significantly lower and cell viability higher in MT2A-overexpressing HeLa cells when ROS production was enhanced by treatment with t-BHP. Mitochondrial membrane potential was noticeably less reduced in both MT-overexpressing cell lines. MT2A overexpression in rotenone-treated cells also significantly reduced or delayed apoptosis induction, as measured by caspase 3/7 activity and cytosolic nucleosome enrichment. We conclude that MT2A offers significant protection against the main death-causing consequences of rotenone-induced complex I deficiency in HeLa cells. Our results are in support of the protective role against oxidative stress ascribed to MTs and provide evidence that MT2A expression may be a beneficial downstream adaptive response in complex I-deficient cells.     

Immunohistochemical localization of (neuro)peptide hormones in endocrine cells and nerves of the gut of a stomachless teleost fish,Barbus conchonius (Cyprinidae)

Summary Enteroendocrine cells containing glucagon-, substance P-, neurotensin- and VIP-like substances have been demonstrated immunocytochemically in the gut of Barbus conchonius. Mainly based on the distribution of the immunoreactive endocrine cells in this and a previous^* study, at least eight different enteroendocrine cell types appear to be present in this stomachless fish: 1. C-terminal-gastrinimmunoreactive cells^*, predominantly present in the upper parts of the folds of the proximal part of the intestinal bulb. 2. Metenkephalin-immunoreactive cells^*, basally located in the folds of the first segment. 3. Pancreatic polypeptide (PP)-immunoreactive cells^*, mainly present in the first half of the first segment. 4. Glucagon-like-immunoreactive (GLI) cells that are basally located in the folds of the first segment and that contain a different polypeptide (possibly glicentin) than pancreatic glucagon cells. 5. Substance P-immunoreactive cells, present in the upper parts of the folds throughout the gut. 6. C-terminal-neurotensin-immunoreactive cells, basally located in the folds throughout the first segment. 7. Vasoactive intestinal polypeptide (VIP)-immunoreactive cells, present in small numbers in the proximal part of the intestinal bulb. 8. Nonspecifically-immunoreactive cells^*, found throughout the intestinal bulb. Many VIP-immunoreactive nerves have been demonstrated in the smooth muscle layer and myenteric plexus of the gut; furthermore some of them are peptide histidineisoleucine (PHI)-immunoreactive. Substance P-, somatostatin-, neurotensin- and met-enkephalin-immunoreactive nerves are also found. Thus, at least partial sequences of four different mammalian neuropeptide hormones (VIP, substance P, neurotensin, met-enkephalin) occur both in endocrine cells and enteric nerves of the gut of B. conchonius.     

The amino-acid sequence of rabbit Cu-Zn superoxide dismutase

The primary structure of Cu-Zn superoxide dismutase from rabbit liver was investigated. The reduced and S-carboxymethylated enzyme was treated with cyanogen bromide, trypsin or Staphylococcus aureus proteinase V8. The resulting peptides were separated by high-performance liquid chromatography and sequenced by automated Edman degradation. With the exception of the N- and C-terminus the complete sequence was established by means of overlapping peptides. The N-terminus is blocked and thus not susceptible to Edman degradation. The amino-acid composition of the tryptic N-terminal peptide corresponds to that of the cytoplasmatic Cu-Zn superoxide dismutases of other mammals investigated. The chromatographic behaviour of these N-terminal peptides on a reversed phase C18 column is also identical, thus suggesting also for the rabbit Cu-Zn superoxide dismutase the N-terminal sequence Ac-Ala-Thr-Lys. The C-terminus was demonstrated to have the sequence -Ile-Ala-Pro by enzymatic degradation with carboxypeptidase Y. The complete amino-acid sequence of the rabbit Cu-Zn superoxide dismutase consists of 152 amino-acids and shows the expected homology to other Cu-Zn enzymes published so far. The aspartate and six histidine residues known to complex the metal ions are conserved at homologous positions. This also applies for the arginine residue near the C-terminus which is supposed to direct the anionic superoxide radical towards the active centre of the enzyme. The amino acid sequence of the rabbit Cu-Zn superoxide dismutase corresponds to those of other mammals in more than 80% of its amino-acid residues. From a total of 152 amino-acid residues the rabbit shares with rat 128, with mouse 130, with horse 127, with pig 126/127, with cattle 130 and with man 131 amino acids in homologous positions. However the Cu-Zn superoxide dismutases of closely related mammals like rats and mice differ in only five amino acid residues of their sequence. A phylogenetic closer relatedness between lagomorphs and rodents than between other orders of mammals, could not be derived from the sequence data given. Rather rodents and lagomorphs are to be considered as two evolutionary independent orders of mammals.     

Oxindole-3-acetic Acid, an Indole-3-acetic Acid Catabolite in Zea mays

A prior study (13) from this laboratory showed that oxidation of exogenously applied indole-3-acetic acid (IAA) to oxindole-3-acetic acid (OxIAA) is the major catabolic pathway for IAA in Zea mays endosperm. In this work, we demonstrate that OxIAA is a naturally occurring compound in shoot and endosperm tissue of Z. mays and that the amount of OxIAA in both shoot and endosperm tissue is approximately the same as the amount of free IAA. Oxindole-3-acetic acid has been reported to be inactive in growth promotion, and thus the rate of oxidation of IAA to OxIAA could be a determinant of IAA levels in Z. mays seedlings and could play a role in the regulation of IAA-mediated growth.     

Immunohistochemical localization of polypeptide hormones in endocrine cells of the digestive tract of Branchiostoma lanceolatum

Summary The digestive tract of the cephalochordate Branchiostoma lanceolatum was investigated with regard to occurrence and distribution of endocrine cells. By the use of the peroxidase-antiperoxidase (PAP) technique, cells in the gut epithelium reacting with antisera against 8 different mammalian polypeptide hormones were localized. Positive reactions were obtained with antisera against the four mammalian islet hormones (insulin, glucagon, pancreatic polypeptide, somatostatin) and against secretin, vasoactive intestinal polypeptide, pentagastrin and neurotensin. No immunoreactivity was found with antisera against members of the lipotropin family (ACTH, met-enkephalin, -endorphin), against big-gastrin, cholecystokinin, substance P and moulin. The exact mapping of the different polypeptide immunoreactive cells throughout the digestive tract of Branchiostoma lanceolatum is presented.     

Changes in polyribosomes and poly(A)-containing polyribosomal RNA in the uterus of the ovariectomized rat in response to oestradiol-17 beta treatment.

Polyribosome formation and the characteristics of polyribosomal poly(A)-containing RNA from uteri of ovariectomized rats responding to a single dose of oestradiol-17 beta was investigated. The mean proportion of polyribosomes in the atrophic uterus was 65%. In response to 10 micrograms of oestradiol-17 beta/100 g body mass, the amount of polyribosomes increased to 88% 24 h after stimulation. Thereafter the proportion of polyribosomes decreased to a value of 48% at 72h. The pattern of amino acid incorporation in oocytes from Xenopus laevis injected with these polyribosomes was similar to the changes in polyribosome formation and degradation. The polyribosomal poly(A)-containing RNA from the controls consisted of a heterogeneous population of RNA with sedimentation values between 5S and 25S. The hormone stimulation resulted in an increase in both the amount and the size (13S to 35S) of the RNA.     

Light- and electron-microscopic analysis of a complex sensory organ: The tegula of Locusta migratoria

Summary Structure and organization of the tegula, a cupola-shaped structure located at the anterior base of the wings of locusts, is described using various morphological methods. Based on histological and cytological criteria, two different sensory systems are distinguished: (1) a field of mechanoreceptive hairs, and (2) a chordotonal organ. The total number of sensory cells corresponds to the number of axons within the nerve supporting the tegula. The hairs are situated at the posterior region of the tegula, and each hair is innervated by only one sensory cell. The complex architecture of the chordotonal organ is analyzed and the attachment of the scolopidia to the cuticle is described. A single scolopidium makes contact with several epidermal cells. The attachment cells run in parallel and are oriented longitudinally within the tegula, being connected to each other and to the epidermal cells by desmosomes. A function in relation to wing movements during flight is suggested for the two sensory systems within the mixed sense organ, tegula.     

Distribution patterns and coexistence of neurohormonal peptides (ANP,BNP, NPY,SP, CGRP,enkephalins) in chromaffin cells and nerve fibers of the anuran adrenal organ

Summary Immunohistochemical techniques were used to study the adrenal organs of the anuran species Rana esculenta, Caldula pulchra and Bufo marinus with respect to the distribution and coexistence of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), substance P (SP), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), Leu-enkephalin (Leu-ENK). Met-enkephalin-Arg-Phe (MEAP) and dynorphin A 1–17 (DYN). Antisera against enzymes involved in catecholamine synthesis, i.e., dopamine--hydroxylase (DBH) and tyrosine hydroxylase (TH), were used for the identification of chromaffin cells. ANP-immunoreactive (-IR) cells occurred in high densities (30%–70% of the total cell population) in all species investigated. In C. pulchra and B. marinus, BNP-IR cells constituted a population of non-DBH-IR and non-TH-IR cells that were different from the ANP-IR cells. A large proportion of the adrenal cells (10%–55%) were immunoreactive to Leu-ENK, and a minority (2%–5%) showed MEAP-immunoreactivity. DYN-immunoreactivity was not observed. The anurans studied exhibited small numbers of SP-IR, CGRP-IR and NPY-IR cells. Immunoreactivities for ANP+Leu-ENK and Leu-ENK+ MEAP were shown to coexist. In C. pulchra and B. marinus, immunoreactions for ANP+NPY, ANP+SP and SP+CGRP were also colocalized. Except for DYN, all neurohormonal peptides also occurred in intra-adrenal nerve fibers. SP-IR fibers also displayed CGRP-immunoreactivity and some Leu-ENK-IR fibers contained MEAP-immunoreactivity. In C. pulchra, NPY-IR fibers were found that also showed ANP-immunoreactivity.Some results of this investigation have been presented in abstract form (Reinecke et al. 1991).     

Occurrence of members of the insulin superfamily in central nervous system and digestive tract of protochordates

Antisera specific for mammalian insulin-like growth factor 1 (IGF-1) and mammalian insulin and the double immunofluorescence technique were used for this study. IGF-1-like-immunoreactivity was localized in entero-endocrine cells in the gastro-intestinal tract of the protochordates Ciona intestinalis and Branchiostoma lanceolatum. Some of the specimens also showed IGF-1-like-immunoreactive (-IR) perikarya and fibers in the central nervous system. Whilst in rat endocrine pancreas, IGF-1-IR and insulin-IR occurred in different cell populations, in Ciona and Branchiostoma the vast majority of entero-endocrine cells and central neurons were IGF-1-like-+insulin-IR. A minor portion exhibited IGF-1-like-IR alone. For further characterization of the IGF-1-like-IR material, in Ciona intestinalis, peptides related to IGF-1 were identified by radioimmunoassay and gel chromatography. In accordance with the immunohistochemical results, IGF-I-like-IR was detected both in cerebral ganglion and in gastro-intestinal tract. Using acid gel chromatography, in Ciona gastro-intestinal tract the IGF-1-like-IR was found to occur in two peaks, with apparent molecular weights of approximately 16 kDa and 3 kDa. Absorption studies with insulin- and IGF-related peptides, with crude extracts and the peak material obtained after gel chromatography, indicated that the IGF-1-like peptides in Ciona are different from mammalian insulin and IGF-1. The findings are in accordance with the presence of a common insulin/IGF precursor molecule in protochordates.